Intrinsically asymmetric cell division, in which a polarized mother cell divides to create daughters with intrinsically different fates, is a fundamental process with critical roles in development, and possible roles in tissue maintenance in the adult life of mammals. To understand asymmetric cell divisions it is necessary to understand the generation and maintenance of cell polarity. Elucidating mechanisms for the establishment and maintenance of cell polarity is an important goal not only because it is central to understanding asymmetric cell division but also because cell polarity is the foundation for the proper development and function of most tissue and organ systems. Failures in cell polarity are a contributing factor in the development of cancer. The proposed research continues ongoing analysis of the generation of cell polarity during the asymmetric divisions of the model organism Caenorhabditis elegans with a particular focus on the role of the PAR proteins in establishing cell polarity in the early embryo. PAR proteins are evolutionarily conserved regulators of cell polarity with roles in a variety of cell types including polarized epithelial cells, neurons, oocytes and migratory cells of many types. Project goals include: 1) Determining the mechanism by which the conserved polarity protein and tumor suppressor LGL-1 restricts PAR proteins to one pole of the cell. 2) Determining the mechanism by which two conserved de-ubiquitinating enzymes regulate the asymmetric localization of the PAR proteins. 3) Using carbon nano-pipettes to develop a microinjection system and using that system to introduce compounds and proteins at specific times and locations in the early embryo to understand the initial events in polarity establishment.